Antibodies, as other protein therapeutics are large and complex molecules and are inherently instable when stored over a period of time, both chemically and physically, potentially resulting in a reduction or loss of activity. Typical chemical instability may result in deamidation, hydrolysis, oxidation, beta-elimination or disulfide exchanges. Physical instability can result in denaturation, aggregation or precipitation.
Therefore, for storage, transport, handling and administration pharmaceutical formulations of antibodies and other proteins have to minimize any of the above phenomena. Antibodies can be formulated in freeze-dried, i.e. lyophilized, form for reconstitution in a solvent shortly before administration, or antibodies can be formulated in liquid form, such as in an aqueous solution. Freeze-dried formulations of antibodies tend to be more stable as water is either a reactant or as a solvent facilitates the transfer of reactants and is thus critical to many routes of chemical degradation that lead to protein instability (Andya, J. D., Hsu, C. C., & Shire, S. J. (2003). Mechanisms of aggregate formation and carbohydrate excipient stabilization of lyophilized humanized monoclonal antibody formulations. AAPS. PharmSci. 5, E10). However, despite the tendency to be less stable, interest has recently focused on liquid formulations of antibodies and other proteins as these are easier and more convenient for the patient and the healthcare professional to handle and administer in comparison to freeze-dried formulations. Liquid formulations do not need to be reconstituted and can be administered with minimal preparation. However, the stabilization of proteins in liquid formulations to avoid or minimize unwanted reactions such as aggregation, precipitation or degradation remains a particular challenge. Aggregation is a particular problem. Individual protein molecules stick physically together resulting, for example, in the formation of insoluble matter or precipitate, which may no longer be active and even cause undesired immunological reactions upon administration. Additionally, a major problem caused by the aggregate formation is that during administration the pharmaceutical formulation may block syringes or pumps.
The suggested physical pathways of degradation to aggregate and particle formation are often described as resulting from a combination of conformational and colloidal stability effects. The conformational stability is the free energy difference between the native folded state and unfolded state under physiological conditions, and as such measures the propensity of an antibody to unfold. Unfolding is often responsible for the overall aggregation rate. Colloidal stability refers to the self-repulsion propensity of native state molecules mainly due to non-specific charges located at the surface area of the molecule.
Aggregation and protein degradation during long term storage are generally assessed by determining the levels of high molecular weight species (HMWS) present in the formulation after a given storage period, typically using size-exclusion chromatography (SEC). Sometimes these events can result in precipitation that is visible to the observer.
In order to be useful liquid pharmaceutical formulations of antibodies and other protein therapeutics need to be long-term stable, and minimize the above reactions in order to contain the correct amount of pharmaceutical ingredient in active form.
Consequently various stabilizers have been explored in the art to help decrease the degradation rates of antibodies via a preferred exclusion mechanism, leading to a layer of excess water surrounding the antibody, and forcing the protein to acquire a more compact state to minimize its surface area. Stabilizers include certain sugars, polyols, amino acids, salts and polymers such as polyethylene glycol. Generally a preferred stabilizer is chosen for a given formulation, although occasionally a combination of stabilizers may be used.
Typically amino acid stabilizers have been employed in liquid antibody formulations for injection, as an alternative to sugars, and generally formulated at a lower pH to optimize antibody stability. Furthermore, glycine is a frequent choice of bulking agent in freeze-dried products where crystallizable compounds are necessary, to act by providing the appropriate texture so as to avoid apparent volume and consistency issues with the formulation such as collapse during the primary drying process. A crystalline bulking agent would act as a filler increasing the density of the solid product and avoiding any risk of structure loss. A crystalline bulking agent also provides homogeneous, dense compositions, it is easy to reconstitute and has a high eutectic temperature allowing for high sublimation temperature during primary drying of the lyophilization process. In this context glycine is used due to its crystallizing, i.e. cryoprotectant, properties, however, once crystallized it no longer has stabilizing capacities, and therefore generally a further stabilizing agent is added to such a formulation.
The choice of stabilizer is also impacted by their respective risk profiles, such as possible effects on blood glucose and their effect on renal function. In this respect, sucrose when administered via an intravenous injection cannot be hydrolyzed and therefore does not affect blood glucose levels. However intravenous antibody products have been associated with renal dysfunction, acute renal failure and osmotic nephrosis, where sugar-stabilized formulations and particularly sucrose, pose the highest risk for acute renal failure due to osmotic nephrosis. Sucrose is a disaccharide comprised of the monosaccharides glucose and fructose, and compared with other organic osmolytes, sucrose is an intermediate-strength protein stabilizer (Street T. O. et al., A molecular mechanism for osmolyte-induced protein stability. Proc. Natl. Acad. Sci. USA, 2006; 103(38):13997-14002).
Additionally formulations usually contain further excipients such as buffering agents, surfactants, or bulking agents (typically present in freeze-dried formulations).
U.S. Pat. No. 8,632,778 discloses a liquid formulation containing a humanized PM-1 antibody in a histidine buffer, containing glycine and being free of sugar.
US 2004/0033228 discloses a liquid formulation comprising an antibody, mannitol, and polysorbate in a citrate or phosphate buffer.
U.S. Pat. No. 6,372,716 discloses a freeze-dried formulation containing factor IX in a histidine buffer containing glycine and sucrose.
Although antibodies have a similar overall structure, they differ in amino acid composition and their glycosylation pattern, as well as possible post-translational modifications such as charge and glycosylation variants. These differences can result in altered interactions with other components that will affect the long term stability of the resulting formulation, which will consequently also require optimization.
With the tendency towards increasing protein concentration within the formulations, to allow for lower administration volumes, the problems regarding protein aggregation and precipitation become more apparent. Given the above, there remains a need in the art to provide further improved liquid pharmaceutical formulations of antibodies with reduced protein aggregation and precipitation after long-term storage.